Characterization and Quantification of Intact 26S Proteasome Proteins by Real-Time Measurement of Intrinsic Fluorescence Prior to Top-down Mass Spectrometry

<div><p>Quantification of gas-phase intact protein ions by mass spectrometry (MS) is impeded by highly-variable ionization, ion transmission, and ion detection efficiencies. Therefore, quantification of proteins using MS-associated techniques is almost exclusively done after proteolysis where peptides serve as proxies for estimating protein abundance. Advances in instrumentation, protein separations, and informatics have made large-scale sequencing of intact proteins using top-down proteomics accessible to the proteomics community; yet quantification of proteins using a top-down workflow has largely been unaddressed. Here we describe a label-free approach to determine the abundance of intact proteins separated by nanoflow liquid chromatography prior to MS analysis by using solution-phase measurements of ultraviolet light-induced intrinsic fluorescence (UV-IF). UV-IF is measured directly at the electrospray interface just prior to the capillary exit where proteins containing at least one tryptophan residue are readily detected. UV-IF quantification was demonstrated using commercially available protein standards and provided more accurate and precise protein quantification than MS ion current. We evaluated the parallel use of UV-IF and top-down tandem MS for quantification and identification of protein subunits and associated proteins from an affinity-purified 26<b> </b>S proteasome sample from <i>Arabidopsis thaliana</i>. We identified 26 unique proteins and quantified 13 tryptophan-containing species. Our analyses discovered previously unidentified N-terminal processing of the β6 (PBF1) and β7 (PBG1) subunit - such processing of PBG1 may generate a heretofore unknown additional protease active site upon cleavage. In addition, our approach permitted the unambiguous identification and quantification both isoforms of the proteasome-associated protein DSS1.</p> </div

UV-IF chromatogram of affinity-purified 20

<p> <b>S proteasome sample from </b><b><i>A. thaliana</i></b><b>.</b> Trp-containing proteins identified using MS/MS with assignment to UV-IF peaks.</p

Kinetic constants determined for SACTE_2347 variants.

a<p>Pure β-1,4 d-mannan.</p>b<p>Acetylated glucomannan contain mannan (60%) and glucose (40%).</p>c<p>Locust bean gum is a natural galactomannan with composition of ∼3.5 mannose per galactose.</p>d<p>IL-pine has the following composition: 34% glucose; 9% xylose; 8% mannose; 4% arabinose, and 8% galactose.</p><p>SACTE_2347 did not hydrolyze cellulose, xylan and other polysaccharides described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094166#s4" target="_blank">Materials and Methods</a>, and likewise did not react with fluorogenic small molecule analogs.</p

Summary of crystal parameters, data collection, and refinement statistics.

<p>*R_merge = ∑_h ∑_i | I_i (h)−|/∑_h∑_i I_i(h), where I_i(h) is the intensity of an individual measurement of the reflection and is the mean intensity of the reflection.</p>§<p>R_cryst = ∑_h ||F_obs|−|F_calc||/∑_h |F_obs|, where F_obs and F_calc are the observed and calculated structure-factor amplitudes, respectively.</p>¶<p>R_free was calculated as R_cryst using 1.5% of randomly selected unique reflections that were omitted from the structure refinement.</p><p>Values in parentheses are for the highest resolution shell.</p

Atomic resolution structure of SACTE_2347.

<p>A, 2Fo-Fc electron density map of eight conserved residues in the GH5 family, contoured at 1.3 σ. Hydrogen atoms were included in the refinement of the high-resolution data. B, Comparison of the active site channels of SACTE_2347 (green) and TfManA (blue). Residues that form the surface of the channel are highlighted in gray, and the positions of loops L1 and L2 are indicated. Positions of the catalytic residues (Glu178 and Glu273 in SACTE_2347_34kDa) are shown in red. Mannobiose observed in the −3 and −2 subsites of the TfManA structure is shown as ball and sticks.</p

Peptide sequences identified by mass spectrometry.

a<p>The full protein sequence of SACTE_2347, annotated with the positions of these peptides is found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094166#pone.0094166.s002" target="_blank">Figure S2</a>. The names of peptides are also used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094166#pone-0094166-g002" target="_blank">Figure 2</a>.</p>b<p>Observed <i>m/z</i>.</p

PCoA clustering of Morisita-Horn Diversity Index.

<p>Sample shape indicates colony. Sample color indicates degradation (<b>A</b>) or layer (<b>B</b>). Panel <b>C</b> shows the correlation analysis. The vectors indicate the correlation of each OTU and the percentage of cellulose degradation with the principal coordinates shown.</p

End products from exhaustive hydrolysis of locust bean gum by SACTE_2347 determined by HPLC.

<p>The three major products identified by comparison of elution times with purified commercial standards were ¹G,²G-M3 (<b>8</b>), ¹G-M2 (<b>5</b>), and M2 (<b>2</b>).</p

Schematic diagram of the binding subsites of SACTE_2347 correlated with reaction of purified oligomannosides and galactosyl-substituted oligomannosides.

<p>The active site schematic shows the positions of sugar binding subsites, the catalytic residues Glu178 and Glu272, and the position of loops L1 and L2. Mannosyl groups (grey circles) and galactosyl groups (black circles) of purified substrates studies are aligned in the −3 to +2 subsites under the schematic of the active site channel. Loop L1 blocks binding of a substituted mannosyl group in either the +1 of +2 subsites. The space between L1 and L2 allows placement of a substituted mannosyl group in the −1 subsite, while shortened L2 allows placement of a substituted mannosyl group into the −2 subsite. All reaction products can be rationalized to arise from hydrolysis of the glycosidic bond between the −1 and +1 subsites after accounting for steric interactions with L1 and L2.</p

Phylogeny of GH5 subfamily 8 including SACTE_2347.

<p>The phylogenic tree was constructed from all sequences assigned to the GH5 subfamily 8. Names shown are GenBank accession codes. Black circles indicate enzymes that have been experimentally verified to exhibit β-mannanase activity; black stars indicate three enzymes whose structures have been determined besides SACTE_2347. These are: <i>Thermomonospora fusca</i>, AAZ54938.1, PDB 1BQC, 2MAN, 3MAN; <i>Bacillus</i> sp. N16-5, AAT06599.1, PDB 2WHJ, 2WJL, 3JUG; <i>Bacillus</i> sp. JAMB-602, BAD99527.1, PDB 1WKY.</p